The present invention relates to the preparation of ring modified cyclic peptide analogs by replacing peptide unit(s) in the cyclic peptide ring nucleus of natural products or semi-synthetic derivatives thereof, in particular, Echinocandin-type compounds, and novel semi-synthetic cyclic peptide compounds produced therefrom.
Echinocandin B is a natural product with antifungal activity that has been modified in the past in a variety of ways. For example, simple derivatives have been made including dihydro-and tetrahydro-reduction products and modification of active groups pendant from the ring nucleus. The most common approach has been replacement of the N-acyl side chain. For example, U.S. Pat. Nos. 4,293,489; 4,320,052; 5,166,135; and 5,541,160; and EP 359529; 448353; 447186; 462531; and 561639 describe a variety of N-acyl derivatized Echinocandin-type compounds that provide varying degrees of antifungal and antiprotozoal activities.
Other modifications have included acylation of the hydroxyl group of the pendant phenolic group. For example, GB 2,242,194; and EP 448343; 448354; 503960 and 525889 describe the introduction of acyl, phosphono and sulfo radicals having a charged group at neutral pH to impart water solubility.
GB 2,241,956 and EP 448355 describe hydrogen-reduction products of cyclohexapeptide compounds.
A review of the Echinocandin families and their semi-synthetic analogs may be found in Turner, W., et al, Current Pharmaceutical Design, 2, 209-224 (1996). The review compares the in vitro and in vivo activities of the Echinocandin natural products and their semi-synthetic analogs.
Each of the approaches described above are limited to reactions with active groups pendant to the cyclic peptide ring nucleus. Some have attempted to build the entire cyclic peptide nucleus synthetically. (See, i.e., U.S. Pat. No. 5,696,084; J. Am. Chem. Soc., 108, 6041 (1986); Evans, D. A., et al., J. Am. Chem. Soc., 109, 5151 (1987); J. Med. Chem., 35, 2843 (1992); and Kurokawa, N., et al., Tetrahedron, 49, 6195 (1993).) However, this approach is not cost effective and may lead to racemic mixtures. Therefore, there is a need to provide a more flexible and cost effective process for modifying the cyclic hexapeptide nucleus of natural products to broaden the scope of potential antifungal candidates.
Several investigators have disclosed the preferential cleavage of an amide bond in compounds bearing hydroxyl groups in the delta and gamma positions relative to the amide bond to provide asymmetric lactones using acids such as hydrochloric acid and trifluoroacetic acid; however, none have applied the process to the cleavage of a terminal amino acid group of a linear peptide. (See, i.e., K. Tanaka, et al., Tetrahedron Lett, 26(10), 1337 (1985); N. Baba, et al., Chem Lett (5), 889 (1989); H. Yoda, et al, Chem Express, 4(8), 515 (1989); and Y. Yamamoto, et al., J Org Chem, 56(3), 112 (1991).)
The present invention provides a method for modifying the cyclic peptide ring system of Echinocandin-type compounds to produce new analogs having antifungal activity. The inventive process allows one to make changes in the cyclic peptide structure of natural and semi-synthetic products that were previously not possible. For example, one may incorporate features such as water-solubility into the cyclic peptide ring nucleus, sites for further modification, increase or decrease the number of amino acid or peptide units within the ring nucleus, and increase or decrease the total number of members (or atoms) in the ring nucleus.
The process includes the steps of (i) providing a cyclic peptide compound comprising a peptide unit having a xcex3-hydroxyl group; (ii) opening the ring of the cyclic peptide compound to provide a first linear peptide wherein the peptide unit having a xcex3-hydroxyl group is the N-terminus peptide unit of the first linear peptide; (iii) cleaving-off the peptide unit having a xcex3-hydroxyl group to provide a second linear peptide (preferably by adding trifluoroacetic acid or hydrochloric acid to the first linear peptide in an organic solvent); (iv) attaching at least one amino acid, a dipeptide unit or a synthetic unit to the second linear peptide to produce a third linear peptide; (v) cyclizing the third linear peptide to produce a modified cyclic peptide compound having a modified ring nucleus. Alternatively, a second peptide unit may be cleaved-off the second linear peptide produced in step (iii) prior to attaching the amino acid, dipeptide or synthetic unit(s) in step (iv) and subsequent cyclization in step (v). The addition of two or more units in step (iv) may be accomplished in a stepwise fashion (e.g., first one unit is attached then a second unit is attached). Of particular interest is the modification of Echinocandin-type compounds to produce novel cyclic hexapeptide and heptapeptide compounds that show inhibition of fungal and parasitic activity. The process also provides a convenient means to produce cyclic peptide compounds having the formulas I and II (including pharmaceutically acceptable salts, esters and hydrates thereof). 
where
R is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or heteroaryl group;
R2 is xe2x80x94H or xe2x80x94CH3;
R3 is xe2x80x94H, xe2x80x94CH3, xe2x80x94CH2CONH2 or xe2x80x94CH2CH2NH2;
R4 is xe2x80x94H or xe2x80x94OH;
R5 is xe2x80x94OH, xe2x80x94OPO3H2, or xe2x80x94OSO3H;
R6 is xe2x80x94H or xe2x80x94OSO3H;
R7 is xe2x80x94CH3 or xe2x80x94H;
(Y) is represented by the following formula 
wherein
A is xe2x80x94(CH2)axe2x80x94 where a 1-4,
xe2x80x94CHRxe2x80x2xe2x80x94CHRxe2x80x3xe2x80x94(CH2)bxe2x80x94, where Rxe2x80x2 and Rxe2x80x3 are independently xe2x80x94H, xe2x80x94OH, C6H5Oxe2x80x94, xe2x80x94SH,
xe2x80x94NH2, CnH2n+1NHxe2x80x94, CnH2n+1Oxe2x80x94, CnH2n+1Sxe2x80x94 or CnH2n+1, where n=1-4 and b=0-1,
xe2x80x94(CH2)cxe2x80x94C(O)NH(CH2)dxe2x80x94, where c=1-2 and d=1-2,
xe2x80x94N=CHxe2x80x94(CH2)exe2x80x94 where e=0-2,
xe2x80x94NRxe2x80x2xe2x80x3(CH2)fxe2x80x94, where Rxe2x80x2xe2x80x3 is xe2x80x94H, xe2x80x94C(O)CH2NH2, xe2x80x94C(O)CH(NH2)CH2NH2 or xe2x80x94CnH2n+1 where n=1-4 and f=1-3,
xe2x80x94(CH2)gxe2x80x94SO2xe2x80x94(CH2)hxe2x80x94 where g=1-2 and h=1-2, 
where j is 1 or 2 and Z is an amino group, alkylamino group, or piperidyl amino group;
B is a substituted or unsubstituted C1 to C6 alkyl group (e.g., isopropyl, p-hydroxybenzyl, hydroxymethyl, or xcex1-hydroxyethyl); and W is a hydrogen or C(O)R where R is as defined above.
In another embodiment of the present invention, novel cyclic peptide compounds are provided having the formulas I and II (above) wherein
A is xe2x80x94(CH2)axe2x80x94 where a=1, 2 or 4,
xe2x80x94CHRxe2x80x2xe2x80x94CHRxe2x80x3xe2x80x94(CH2)bxe2x80x94 where Rxe2x80x2 and Rxe2x80x3 are independently xe2x80x94H, xe2x80x94OH, C6H5Oxe2x80x94, xe2x80x94SH,
xe2x80x94NH2, CnH2n+1NHxe2x80x94, CnH2n+1O, CnH2n+1Sxe2x80x94or CnH2n+1, where n=1-4 and b=0,
xe2x80x94(CH2)cxe2x80x94C(O)NH(CH2)dxe2x80x94 where c=1-2 and d=1-2,
xe2x80x94N=CHxe2x80x94(CH2)xe2x80x94 where e=0-2,
xe2x80x94NRxe2x80x2xe2x80x3(CH2)fxe2x80x94 where Rxe2x80x2xe2x80x3 is xe2x80x94H, xe2x80x94C(O)CH2NH2, xe2x80x94C(O)CH(NH2)CH2NH2 or where n=1-4 and f=1-3,
xe2x80x94(CH2)xe2x80x94SO2xe2x80x94(CH2)hxe2x80x94 where g=1-2 and h=1-2, 
where j is 1 or 2 and Z is an amino group, alkylamino group, or piperidylamino group.
In yet another embodiment of the present invention, a pharmaceutical composition is provided comprising the novel compounds I and II described above (including pharmaceutically acceptable salts, esters and hydrates thereof) in a pharmaceutically inert carrier. Methods for using the novel compounds and pharmaceutical compositions described above for inhibiting fungal growth and parasitic activity are also provided, as well as a method for treating a fungal infection in a human comprising administering to a human in need of such treatment a therapeutically effective amount of the novel antifungal compound described above.
As used herein, the term xe2x80x9cEchinocandin-type compoundsxe2x80x9d refers to compounds having the following general structure including any simple derivatives thereof: 
wherein R is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or heteroaryl group; R1 is xe2x80x94H or xe2x80x94OH; R2 is xe2x80x94H or xe2x80x94CH3; R3 is xe2x80x94H, xe2x80x94CH3, xe2x80x94CH2CONH2 or xe2x80x94CH2CH2NH2;
R4 is xe2x80x94H or xe2x80x94OH; R5 is xe2x80x94OH, xe2x80x94OPO3H2, or xe2x80x94OSO3H; and R6 is xe2x80x94H or xe2x80x94OSO3H.
The term xe2x80x9calkylxe2x80x9d refers to a hydrocarbon radical of the general formula CnH2n+1 containing from 1 to 30 carbon atoms unless otherwise indicated. The alkane radical may be straight, branched, cyclic, or multi-cyclic. The alkane radical may be substituted or unsubstituted. Similarly, the alkyl portion of an alkoxy group or alkanoate have the same definition as above.
The term xe2x80x9calkenylxe2x80x9d refers to an acyclic hydrocarbon containing at least one carbon-carbon double bond. The alkene radical may be straight, branched, cyclic, or multi-cyclic. The alkene radical may be substituted or unsubstituted. The term xe2x80x9calkynylxe2x80x9d refers to an acyclic hydrocarbon containing at least one carbon-carbon triple bond. The alkyne radical may be straight, or branched. The alkyne radical may be substituted or unsubstituted.
The term xe2x80x9carylxe2x80x9d refers to aromatic moieties having single (e.g., phenyl) or fused ring systems (e.g., naphthalene, anthracene, phenanthrene, etc.). The aryl groups may be substituted or unsubstituted. Substituted aryl groups include a chain of aromatic moieties (e.g., biphenyl, terphenyl, phenylnaphthalyl, etc.)
The term xe2x80x9cheteroarylxe2x80x9d refers to aromatic moieties containing at least one heteratom within the aromatic ring system (e.g., pyrrole, pyridine, indole, thiophene, furan, benzofuran, imidazole, pyrimidine, purine, benzimidazole, quinoline, etc.). The aromatic moiety may consist of a single or fused ring system. The heteroaryl groups may be substituted or unsubstituted.
Within the field of organic chemistry and particularly within the field of organic biochemistry, it is widely understood that significant substitution of compounds is tolerated or even useful. In the present invention, for example, the term alkyl group allows for substituents which is a classic alkyl, such as methyl, ethyl, propyl, hexyl, isooctyl, dodecyl, stearyl, etc. The term group specifically envisions and allows for substitutions on alkyls which are common in the art, such as hydroxy, halogen, alkoxy, carbonyl, keto, ester, carbamato, etc., as well as including the unsubstituted alkyl moiety. However, it is generally understood by those skilled in the art that the substituents should be selected so as to not adversely affect the pharmacological characteristics of the compound or adversely interfere with the use of the medicament. Suitable substituents for any of the groups defined above include alkyl, alkenyl, alkynyl, aryl, halo, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, mono- and di-alkyl amino, quaternary ammonium salts, aminoalkoxy, hydroxyalkylamino, aminoalkylthio, carbamyl, carbonyl, carboxy, glycolyl, glycyl, hydrazino, guanyl, and combinations thereof.
The synthetic scheme outlined below illustrates the general procedures for modifying the cyclic peptide ring system of Echinocandin-type compounds while maintaining chirality. The cyclic peptide ring of any Echinocandin-type natural product or semi-synthetic derivative can be opened and the terminal ornithine peptide unit cleaved so long as the xcex3-hydroxyl group of the ornithine peptide unit is present and not blocked. The term xe2x80x9cnatural productxe2x80x9d refers to those secondary metabolites, usually of relatively complex structure, which are of more restricted distribution and more characteristic of a specific source in nature. Suitable natural product starting materials belonging to the Echinocandin cyclic peptide family include Echinocandin B, Echinocandin C, Aculeacin Axcex3, Mulundocandin, Sporiofungin A, Pneumocandin A0, WF11899A, and Pneumocandin B0. For illustrative purposes, the following synthetic scheme starts with Cilofungin. 
As shown above, the cyclic hexapeptide ring (1) is first opened using base catalysis and then reduced with sodium borohydride to give the linear hexapeptide (2). Upon treatment with triethyl silane in trifluoroacetic acid (TFA), the benzylic hydroxyl is removed and the ornithine unit is cleaved to give the linear pentapeptide (3). The linear pentapeptide (2) can now be protected and the primary amide activated to provide an intermediate (4) which can be recyclized with a new amino acid unit or other synthetic unit to produce a new cyclic compound (5). Cyclic compound (5) can be further modified by deprotecting and acylating the pendant amino group (if present) to provide modified cyclic compound (6) having an N-acyl side chain. Those skilled in the art will appreciate that the N-acyl side chain encompasses a variety of side chain moieties known in the art. Suitable side chain moieties include substituted and unsubstituted alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups and combinations thereof. Preferably, the side chain contains both a linearly rigid section and a flexible alkyl section to maximize antifungal potency. In addition, further modifications can be made on any new functionality introduced by the incorporation of the new amino acid, peptide or synthetic unit(s) containing such new functionality.
Alternatively, another peptide unit can be cleaved from intermediate (3) to provide a tetrapeptide (7) which can be recyclized with a new amino acid unit, dipeptide unit, or other synthetic unit to produce a new cyclic compound (8). Like cyclic compound (5), compound (8) can also be further modified by deprotecting and acylating the pendant amino group (if present) to attach an N-acyl side chain or modification of any new functionality introduced through the incorporation of the new amino acid, peptide or synthetic units.
As illustrated in the synthetic scheme above, the ring nucleus is selectively opened at the C-terminus L-proline, N-terminus R-omithine linkage using standard base catalysis well known to those skilled in the art. Once the cyclic hexapeptide is open, then the terminal omithine amino acid may be cleaved and a new amino acid (or other synthetic unit) attached using standard peptide formation processes (or condensation processes) well known to those skilled in the art. The terminal ornithine unit is cleaved with trifluoroacetic acid or hydrochloric acid in an organic solvent such as methylene chloride, toluene, or dioxane. The preferred reaction condition is trifluoroacetic acid in methylene chloride.
Any amino acid or peptide unit may be attached to the linear peptide. Theoretically, it is also possible to condense other synthetic units onto the peptide that are capable of cyclization. For example, a sulfonamide linkage may be formed between a terminal amino group on the linear peptide and a sulfonyl group on a synthetic unit. Any number of other linkages may also be envisioned; however, the pharmaceutical activity of such compounds are currently unknown.
The insertion of a new amino acid, dipeptide unit or other synthetic unit allows one to change the size of the cyclopeptide ring. The number of atoms in the ring system may be increased or decreased from the original 21 membered Echinocandin ring structure depending upon the particular compound(s) inserted into the ring. Theoretically, the ring size is limited only by the configuration of the linear peptide. If the linear peptide is too short or too long, the ends cannot come into close enough proximity to react and the ends may polymerize with another linear peptide rather than close to form a ring. The optimum configuration of the linear peptide for ring closure will vary depending upon the particular amino acids that make-up the peptide structure. For Echinocandin-type compounds, preferably, the final ring structure contains between 19 to 22 members, more preferably, the final ring structure is a 21- or 22-membered ring, most preferably the final ring structure is a 21-membered ring.
It is well-known that the acyl side chain pendant from the Echinocandin ring structure plays an important role in the activity of both the natural products and semi-synthetic Echinocandin type materials. Consequently, any amino acid or synthetic unit may be used for insertion into the ring so long as the final cyclized product contains at least one amino group capable of acylation.
When the inserted compound contains more than one unit, the units may be attached one at a time to the linear penta- or tetra-peptide or the individual units can be combined and then added to the linear penta- or tetra-peptide as a block unit. Preferably, the units are added as a block unit to minimize racemization.
Acylation of the amino group may be accomplished in a variety of ways well known to those skilled in the art. For example, the amino group may be acylated by reaction with an appropriately substituted acyl halide, preferably in the presence of an acid scavenger such as a tertiary amine (e.g., triethylamine). The reaction is typically carried out at a temperature between about xe2x88x9220xc2x0 C. to 25xc2x0 C. Suitable reaction solvents include polar aprotic solvents, such as dioxane or dimethylformamide. Solvent choice is not critical so long as the solvent employed is inert to the ongoing reaction and the reactants are sufficiently solubilized to effect the desired reaction.
The amino group may also be acylated by reaction with an appropriately substituted carboxylic acid, in the presence of a coupling agent. Suitable coupling agents include dicyclohexylcarbodiimide (DCC), N,Nxe2x80x2-carbonyldiimidazole, bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), benzotriazole-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and the like.
Alternately, the amino group may be acylated with an activated ester of a carboxylic acid such as p-nitrophenyl, 2,4,5-trichlorophenyl, hydroxybenzotriazole hydrate (HOBT.H2O), pentafluorophenol, and N-hydroxysuccinimide carboxylate esters. Preferred acylating moieties are the 2,4,5-trichlorophenyl and HOBT carboxylate esters. The reaction is typically ran 1 to 65 hours at a temperature from about 0xc2x0 C. to 30xc2x0 C. in an aprotic solvent. The reaction is generally complete after about 24 to 48 hours when carried out at a temperature between about 15xc2x0 C. to 30xc2x0 C. Suitable solvents include tetrahydrofuran and dimethylformamide or mixtures thereof. The amino group is generally present in equimolar proportions relative to the activated ester or with a slight excess of the amino group.
The compounds of the present invention may be isolated and used per se or in the form of its pharmaceutically acceptable salt or hydrate. The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d refers to non-toxic acid addition salts derived from inorganic and organic acids. Suitable salt derivatives include halides, thiocyanates, sulfates, bisulfates, sulfites, bisulfites, arylsulfonates, alkylsulfates, phosphonates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphonates, alkanoates, cycloalkylalkanoates, arylalkonates, adipates, alginates, aspartates, benzoates, fumarates, glucoheptanoates, glycerophosphates, lactates, maleates, nicotinates, oxalates, palmitates, pectinates, picrates, pivalates, succinates, tartarates, citrates, camphorates, camphorsulfonates, digluconates, trifluoroacetates, and the like.
The ring-modified compounds may be used in a variety of pharmaceutical formulations. A typical formulation comprises the ring-modified compound (or its pharmaceutically acceptable salt, ester or hydrate) in combination with a pharmaceutically acceptable carrier, diluent or excipient. The active ingredient is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product. Formulations may comprise from 0.1% to 99.9% by weight of active ingredient, more generally from about 10% to about 30% by weight.
As used herein, the term xe2x80x9cunit dosexe2x80x9d or xe2x80x9cunit dosagexe2x80x9d refers to physically discrete units that contain a predetermined quantity of active ingredient calculated to produce a desired therapeutic effect. When a unit dose is administered orally or parenterally, it is typically provided in the form of a tablet, capsule, pill, powder packet, topical composition, suppository, wafer, measured units in ampoules or in multidose containers, etc. Alternatively, a unit dose may be administered in the form of a dry or liquid aerosol which may be inhaled or sprayed.
The dosage to be administered may vary depending upon the physical characteristics of the patient, the severity of the patient""s symptoms, and the means used to administer the drug. The specific dose for a given patient is usually set by the judgment of the attending physician.
Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like. The particular carrier, diluent or excipient used will depend upon the means and purpose for which the active ingredient is being applied. The formulations may also include wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, sweeteners, stabilizers, perfuming agents, flavoring agents and combinations thereof.
A pharmaceutical composition may be administered using a variety of methods. Suitable methods include topical (e.g., ointments or sprays), oral, injection and inhalation. The particular treatment method used will depend upon the type of infection being addressed.
Echinocandin-type compounds have been shown to exhibit antifungal and antiparasitic activity such as growth inhibition of various infectious fungi including Candida spp. (i.e., C. Albicans, C. Parapsilosis, C. Krusei, C. Glabrata, C. Tropicalis, or C. Lusitaniaw); Torulopus spp.(i.e., T. Glabrata); Aspergillus spp. (i.e., A. Fumigatus); Histoplasma spp. (i.e., H. Capsulatum); Cryptococcus spp. (i.e., C. Neoformans); Blastomyces spp. (i.e., B. Dermatitidis); Fusarium spp.; Trichophyton spp., Pseudallescheria boydii, Coccidioides immits, Sporothrix schenckii, etc.
Compounds of this type also inhibit the growth of certain organisms primarily responsible for opportunistic infections in immunosuppressed individuals, such as growth inhibition of Pneumocystis carinii (the causative organism of pneumocystis pneumonia (PCP) in AIDS and other immunocompromised patients. Other protozoans that are inhibited by Echinocandin-type compounds include Plasmodium spp., Leishmania spp., Trypanosoma spp., Cryptosporidium spp., Isospora spp., Cyclospora spp., Trichomnas spp., Microsporidiosis spp., etc.
The compounds of the present invention are useful in combating either systemic fungal infections or fugal skin infections. Accordingly, a method is provided for inhibiting fungal activity comprising contacting a compound of formula I or II (or a pharmaceutically acceptable salt, ester or hydrate thereof) with a fungus. A preferred method includes inhibiting Candida albicans or Aspergillus fumigatis activity. The term xe2x80x9ccontactingxe2x80x9d includes a union or junction, or apparent touching or mutual tangency of a compound of the invention with a parasite or fungus. The term does not imply any further limitations to the process, such as by mechanism of inhibition. The methods are defined to encompass the inhibition of parasitic and fungal activity by the action of the compounds and their inherent antiparasitic and antifungal properties.
A method for treating a fungal infection which comprises administering an effective amount of a compound of formula I or II (or a pharmaceutically acceptable salt, ester or hydrate thereof) to a host in need of such treatment is also provided. A preferred method includes treating a Candida albicans or Aspergillus fumigatis infection. The term xe2x80x9ceffective amountxe2x80x9d refers to an amount of active compound which is capable of inhibiting fungal activity. The dose administered will vary depending on such factors as the nature and severity of the infection, the age and general health of the host and the tolerance of the host to the antifungal agent. The particular dose regimen likewise may vary according to these factors. The medicament may be given in a single daily dose or in multiple doses during the day. The regimen may last from about 2-3 days to about 2-3 weeks or longer. A typical daily dose (administered in single or divided doses) contains a dosage level between about 0.01 mg/kg to 100 mg/kg of body weight of an active compound. Preferred daily doses are generally between about 0.1 mg/kg to 60 mg/kg and more preferably between about 2.5 mg/kg to 40 mg/kg.
Although the compounds described herein may be used for inhibiting fungal and parasitic activity in a variety of circumstances (e.g., humans, animals, agriculture, etc.), preferably, the methods of use are limited to the treatment of humans to reduce the potential for developing resistance to the pharmaceutical.